Alternative pre-mRNA splicing affects the expression of gene products involved in many aspects of cell growth and differentiation, yet the mechanisms by which it is regulated are only beginning to be elucidated. SR proteins and related factors are thought to play important roles in the regulation of splicing. These factors form regulatory complexes at sites internal to the exons of alternatively spliced pre-mRNAs and function to recruit factors from the general splicing machinery to nearby splice sites that otherwise would not be recognized. Although SR proteins are highly phosphorylated, the role of phosphorylation in the regulation of splicing is largely unknown. We propose to investigate how SR protein phosphorylation affects the assembly and function of sex-specific regulatory complexes formed on a splicing enhancer in the doublesex pre-mRNA. Preliminary studies indicate that the LAMMER kinase Darkener-of- apricot phosphorylates SR proteins and is required for normal regulation of doublesex RNA splicing. We will test whether the phosphorylation of proteins from the doublesex splicing enhancer complex affects the ability of SR proteins, and related regulatory factors such as TRA and TRA2, to interact with each other and with the pre-mRNA. Other experiments will compare the effects of different classes of kinases on SR protein function. Finally, we will produce strains of Drosophila with mutations in SR protein kinases to examine their roles in development and on the phosphorylation of specific SR proteins in vivo. We anticipate that these studies will provide significant new insights into the mechanisms by which splicing is regulated and will open up new avenues for future studies on phosphorylation of SR proteins.